As used herein, the term "cytokine" is defined as growth factors secreted by immune or other cells, whose action is on cells of the immune system, such as, but not limited to, T-cells, B-cells, NK cells and macrophages. Representative cytokines include, but are not limited to, the group consisting of interleukin 1.alpha., interleukin-1.beta., interleukin-2, interleukin-6, interferon-alpha, interferon-gamma, tumor necrosis factor-.alpha., growth factors, such as TGFB, NGF, EGF, and oncogenes such as c-myc and c-fos. The term "EIA" means any immunoassay utilizing enzymes as the label. The term "endogenous cytokines" as used herein, means cytokines that are produced in vivo and normally circulate in the blood and various other biological fluids. The term includes prohormones which are larger molecular weight forms of cytokines which have not yet undergone post-transcriptional modification.
The impact of a variety of factors, including behavioral and environmental stress, on health and on susceptibility to diseases such as AIDS, cancer and autoimmune disease, is thought to be mediated by the immune system (Ader, R. et al., eds., PSYCHONEUROIMMUNOLOGY, Second Edition, Academic Press, New York (1991)). However, the effects of many of these influences on the function of the immune and host defense systems has been difficult to assess with certainty. This is due, in part, to the fact that most methods of evaluating the activity of the immune system focus on blood components, whether serum or cells.
A newly emerging field known as psychoneuroimmunology seeks to define, in a more mechanistic manner, the way in which biobehavioral factors are internally transduced to impact on the immune system and thereby influence susceptibility or resistance to a variety of pathological processes. Biobehavioral factors such as stress may be positive or negative risk factors for a pathologic outcome (Maier, S. F. et al., BRAIN BEHAV. IMMUN. 2: 8791 (1988)). It is thought by some researchers that a positive or negative attitude in humans may be a significant factor contributing to the overall host response in cancer, AIDS, or autoimmunity (see, for example, Ader et al., supra).
Our ability to evaluate and quantify discrete components of the immune system in a single individual over time (critical for monitoring the onset and/or treatment of diseases associated with AIDS, for example) has been elusive (Kiecolt et al., BRAIN BEHAV. IMMUN. 2: 67-78 (1988); Glaser, R. et al., BRAIN BEHAV. IMMUN. 1: 107-112 (1987)). The study of behavioral strategies that can be enlisted in the modulation of the immune system requires readily available analytical tools that can be used by a broad spectrum of investigators, including those not necessarily sophisticated in complex in vitro immunological techniques. Such methods are clearly lacking at present.
One method used widely in psychoneuroimmunology research is the stimulation of lymphocytes in vitro by mitogens, known also as lymphocyte blastogenesis or the lymphocyte transformation test (Maluish, A. E. et al., MANUAL OF CLINICAL LABORATORY IMMUNOLOGY (3rd Ed.), Rose, N. R., et al., (eds.), 274-281, (1986)). This method involves culture of lymphocytes separated from blood for several days in vitro in the presence of varying doses of mitogens. These mitogens are typically plant-derived or microorganism-derived non-specific activators of lymphocyte -proliferation. The ability of the cultured lymphocytes to incorporate .sup.3 H-thymidine (a well-known measure of DNA synthesis and cell growth) is generally used as a measure of the cells' response. This procedure is highly variable and technician-dependent, and is influenced by a number of unknown factors including the microenvironment provided by the subject's serum if incorporated in the assay.
In contrast to the more classic immunoassays for endocrine hormones, the absence of an established baseline response makes comparison between laboratories, and even within a laboratory, very difficult. However, the lymphocyte transformation test does provide functional information about the immune system because the test examines the response capacity of an entire class of cells, either T or B lymphocytes, depending upon the mitogen used. Unfortunately, the imprecision of the assay greatly diminishes its utility.
A second assay commonly used in psychoneuroimmunology research measures natural killer (NK) cell activity (Herberman, R. B., MANUAL OF CLINICAL LABORATORY IMMUNOLOGY (3rd Ed.), Rose, N. R., et al. (eds.), 308-314 (1986); Irwin, M. et al., BRAIN BEHAV. IMMUN. 1: 98-104 (1987); Jemmott, J. B. 3d, J. BEHAV. MED. 13: 53-73 (1990)). Again, this assay suffers from many of the shortcomings noted above. One reason this assay has become popular is the relative ease of its performance compared to most antigen-specific or antibody-dependent cellular cytotoxicity assays. However, compared to immunoassays commonly used for endocrine hormones, it still ranks as a rather difficult bioassay to perform.
The NK cell assay is based on the ability of a class of white blood cells to spontaneously lyse target cells, typically from an appropriately sensitive tumor cell line, which have been pre-labeled with .sup.51 Cr, thereby releasing the radioisotope into the medium. This assay lacks a quantifiable baseline, and comparisons between individuals is based on the differences in lysis effected by serial dilutions of a blood cell sample. The precise relationship between cells with NK function and the better known classes of leukocytes and lymphocytes is still unclear. Furthermore, the role of NK cells in immunological function and host defense to cancer or infectious disease is not firmly established.
A third approach to monitoring the immune system that has grown in popularity is the enumeration of lymphocyte subsets by flow cytometry using antibodies specific for cell-surface markers (Ault, K., MANUAL OF CLINICAL LABORATORY IMMUNOLOGY (3rd Ed.), Rose, N. R., et al., (eds.), 247-253 (1986)). This approach provides a snapshot of the distribution of various lymphocyte classes in the circulation, but does not provide functional information about any of the cells. Because many psychobiological studies involve a pre-treatment, treatment and post-treatment design, s the assay suffers from the disadvantage that some changes in cell numbers may be too rapid and transient to be meaningful. In contrast, the long-term status of circulating lymphocyte subsets may be too stable to provide an accurate measure of a response to a mild stimulus. The approach of enumerating subsets is thus more appropriate for examination of more permanent changes that occur in major medical illnesses, such as the loss of CD4.sup.+ T cells in AIDS. Therefore, the utility of this method for measuring the response of the immune system to behavioral factors is limited.
A fourth approach, which has the distinct advantage of simple and non-stressful sampling, involves measurement of the secretory immunoglobulin, IgA, in the saliva (Johnson R. B., Jr. et al., J. IMMUNOASSAY 3: 73-89 (1982); Stone, A. A. et al., J. HUMAN STRESS 13: 136-140 (1987); Jemmott, J. B. 3d et al., BEHAV. MED. 15: 63-71 (1989); Jemmott, J. B. 3d et al., J. PERS. SOC. PSYCHOL. 55: 803-810 (1988); Jemmott, J. B. 3d et al., LANCET 1: 1400-1402 (1983)). The total concentration of salivary IgA reflects the presence of a large collection of antibodies of unknown antigen specificity. Furthermore, it is not clear how total IgA levels in saliva relate to the overall dynamics of the immune system. IgA antibodies are associated with mucosal surfaces and are thought to protect these surfaces from infection. Consequently, secretory IgA is found not only in saliva but also in tracheobronchial secretions, colostrum, milk and genitourinary secretions. The utility of monitoring secreted IgA as a useful index of activity of the immune system as a whole has been questioned (Stone et al., supra; Jemmott et al., supra).
Importantly, for testing behavioral factors, such as psychological stress on the immune system, it is particularly important to be able to sample a body fluid in a non-stressful manner. For example, obtaining a blood sample by venipuncture may itself induce physiological changes which could contaminate the data obtained. Thus, a method which would allow measurement of salivary levels of a cytokine or other product of the immune system would have several major advantages over existing approaches, such as (a) eliminating the risk and stress associated with phlebotomy; (b) serving as a window into the internal milieu; (c) allowing simple "at home" collection; (d) permitting a dynamic assessment over time; (e) providing an essential new tool for the evaluation of brain/immune system interactions; (f) serving as a measure which will assist in evaluating the impact of stress on health and (g) providing a measure that may be relevant to the onset of disease after initial pathogenic events, such as HIV infection in AIDS.
Two regulatory molecules of the immune system are interleukin 1 and 2 (IL-1 and IL-2). The ability of IL- 1 and IL-2 to modulate a cytokine "cascade" and the concomitant cell proliferation, differentiation and effector function of lymphoid cells has been characterized in detail (see, for example, Kampschmidt, R. F., J. LEUK. BIOL. 36: 341-355 (1984); Smith, K. A., ANN. REV. IMMUNOL. 2: 319-333 (1984)).
These two molecular signals are apparently not limited to action within the immune system, as recent studies indicate that both of these cytokines act as homeostatic regulators outside the immune system. For example, IL-1 has been shown to act as a potential modulator of the hypothalamic-pituitary-adrenal axis (Besedovsky, H. et al., SCIENCE 233: 652-654 (1986); Bernton, E. W. et al., SCIENCE 1987: 519-521 (1987)), while IL-2 has been shown to affect Leydig cell steroidogenesis (Gou, H. et al., ENDOCRINOLOGY 127: 1234-1239 (1990)). Studies have demonstrated that IL-1 and IL-2 directly (without the presence of macrophages or T-cells) inhibited the in vitro and in vivo growth of hormone-dependent human breast cancer cells (Paciotti, G. F. et al., MOL. ENDOCRINOL. 2: 459-464 (1988); Paciotti, G. F. et al., ANTICANCER RES. 8: 1233-1240 (1988); Paciotti, G. F. et al., ANTICANCER RES. 11: 25-32 (1991)). It appears, therefore, that these cytokines not only affect classical autocrine/paracrine loops within the immune system, but also affect endocrine circuits and may therefore modulate interactions between the endocrine and immune systems.
For IL-1 and IL-2 to play roles as "endocrine-like" hormones, they must exist in sufficient quantities to reach and affect their hypothesized target sites. Thus, ascertaining the biological basis of an endocrine action for such signal molecules traditionally thought to belong to the immune system, depends on our ability to detect and monitor, in a quantitative manner, the endogenous concentrations of these molecules. Conventional methods of measuring IL-1 and IL-2, as well as other cytokines, directly in the blood have been unsatisfactory.
To date, the study of IL-1 and IL-2 has been concerned primarily with their role in stimulating lymphocyte proliferation and helper and effector function in the adaptive immune response and other forms of host defense. As a result of this focus, study of these cytokines has been limited to stimulation of cells cultured in vitro, or the measurement of blood cytokine levels in vivo in immunological diseases. Recent studies indicate that IL-1 should be considered an "endogenous" component of the circulation; its concentration can vary in a number of "real life" situations.
Generally, in viewing IL-1 and IL-2 or other cytokines as measures of immunocompetence in vivo, investigators have focused on large changes in concentration under severe pathophysiological conditions such as leukemia and arthritis. Furthermore, reports of circulating cytokine levels have been concerned with the elevation in patients compared with controls, while paying little attention to the fact that IL-1 and IL-2 were also detectable in normal subjects and might be subject to modulation by a variety of subclinical factors (Michie, H. R. et al., NEW ENG. J. MED. 318: 1481-1486 (1988); Grau, G. E. et al., LYMPHOKINE RES. 7: 335 (1988); Shenkin, A. et al., LYMPHOKINE RES. 7, 333 (1988)).
In addition to measurement of cytokines in serum or plasma, various cytokines have been detected in other biological fluids. For example, Kimball, E. C. et al., (J. IMMUNOL. 133: 256-260 (1984)) reported IL-1 bioactivity in human urine. Tamatani, T. et al., (IMMUNOLOGY 65: 337-342 (1988)) disclosed the presence of IL-1.alpha. and IL-1.beta. in human amniotic fluid, using chromatographic and bioassay methods. The same group used enzyme immunoassays to measure IL-1.alpha. and IL-1.beta. in human amniotic fluid (Tsunoda, H. et al., LYMPHOKINE RES. 7: 333 (1988)). Wilmott, R. W. et al., (LYMPHOKINE RES. 7: 334 (1988)) measured IL-1.beta. (by EIA) and IL-1 bioactivity in human bronchoalveolar lavage fluid in cystic fibrosis compared to other diseases. Khan, et al. (MOL. CELL. ENDOCRINOL. 58: 221-230 (1988)) reported that high levels of IL-1-like bioactivity could be demonstrated in human ovarian follicular fluid. Lymphotoxins have been reported in blister fluid of pemphigoid patients (Jeffes, E. W. et al., J. CLIN. IMMUNOL. 4: 31-35 (1984)). IL-1 has also been reported in human sweat (Didierjean, et al., "Biologically active interleukin 1 in human eccrine sweat: Site dependent variations in .alpha./.beta. ratios and stress-induced increased secretion," CYTOKINE 2: 438-446 (1990)).
IL-1 has been reported to be found in the cerebrospinal fluid (CSF) of cats (Coceani, F. et al., BRAIN RES. 446: 245-250 (1988)) and humans (see, for example, Peter, J. B. et al., NEUROLOGY 41: 121-123 (January 1991)). When Peter et al. (supra) examined IL-1.beta. and tumor necrosis factor (TNF) in CSF and serum of multiple sclerosis patients and normal controls, they concluded that the levels of these cytokines in these two fluids were not of prognostic or diagnostic utility. Westacott, C. I. et al., (ANN. RHEUM. DIS. 49: 676-681 (1990)) used immunoassays to measure cytokines in synovial fluid of patients with rheumatic disease (EIA for IL-1.beta.; RIA for IL-2, TNF, IFN alpha and gamma).
A factor with IL-1-like bioactivity was detected in the gingival fluid of clinically normal humans (Oppenheim, J. J. et al., TRANSPLANT. PROC. 14: 553-555 (1982)), the activity being higher in inflamed than non-inflamed gingival regions. The gingival fluid factor exhibited molecular weights corresponding to both IL- I and epidermal thymocyte-activating factor (Charon, J. A. et al., INFEC. IMMUN. 38: 1190-1195 (1982)). Studies by Jandinski and colleagues using EIA (Jandinski, J. et al., J. DENT. RES. 67: 2307 (1988); Jandinski, J. et al., J. DENT. RES. 68: 526 (1988); Jandinski, J. et al.. J DENT. RES. 68: 1233 (1988)) reported the presence of IL-1.alpha. in periodontal tissue, while IL-1 predominated in gingival crevicular fluid of patients with periodontal disease. A more recent study (Kabashimi, H. et al., INFEC. IMMUN. 58: 2621-2627 (1990)) utilizing polyclonal antisera to recombinant human IL-1.alpha. and IL-1.beta. and measurement by Western blotting, disclosed that the majority of the IL-1 bioactivity found in gingival crevicular fluid of patients with chronic inflammatory periodontal disease was IL-1, generally considered the membrane-bound form of IL-1. The suggestion was made that the IL-1 was derived by enzymatic cleavage from the cell surface. In this latter study, special care was taken to avoid contamination of the gingival fluid with saliva. These facts argue strongly against a salivary origin for the gingival fluid IL-1.
As can be seen from the foregoing overview of the literature, there have been many attempts to measure endogenous cytokines in blood and other body fluids. However, in reviewing these reports, it is apparent that there is wide variation in the reported results with regard to cytokine concentration in the blood and to fluctuations of cytokine concentration in the blood.
Many reports indicate that cytokines (i.e., IL-2) are not detectable in normal subjects using immunoassays. It is possible that circulating IL-2 may be bound by the well-described soluble IL-2 receptor. The site of attachment may interfere with recognition by the capture antibody of a sandwich assay system, which would make IL-2 appear undetectable. Alternatively, even if the molecule is captured, the detection by the second antibody may be prevented by steric hindrance by the binding of both the capture antibody and the soluble IL-2 receptor to IL-2. In effect, there may not be sufficient space to permit the binding of yet a third large protein. The key element is that the design of these sandwich ELISA assays suggests that they may only pickup a fraction of the total.
It is possible that some assay procedures detect very little cytokine, whereas others pickup none at all. This difference may be related to the assay system or to the cytokine or both. The problem has been reported by the observations of Cannon, J. G., et al., LYMPHOKINE RES., 7: 457-465 (1988), in which the authors show that some plasma substance inhibited the assay, effecting detection. In this study, the authors recommend chloroform extraction of plasma to remove interfering substances. It is not clear from this study if the plasma factors simply effect the performance of the assay or are related to the cytokine itself. This question was further described by Capper, S. J., et al., CYTOKINE 2: 182-189 (1990). Capper, S. J., et al. show that IL-1 .alpha. and .beta. are bound by proteins and that the dissociation of these molecules from these serum binding proteins by acidifying the plasma changes the detectable levels. The problem of masking appears to be unique to biological samples collected from in vivo sources; in vitro cell culture supernatants do not contain large quantities of serum binding proteins.
The best example of a cytokine "binding protein" may be inferred from the data describing a soluble IL-2 receptor found in the circulation. This molecule has been shown to be immunologically similar to the low affinity IL-2 receptor on T-cells, "Tac". Its presence in the circulation, free of the T-cell, would strongly argue that it can no longer be a receptor capable of transmitting signals from outside the cell to inside the cell. However, these data do not address the ability of this molecule to still bind IL-2. We would argue that IL-2 in the circulation is bound by this protein, and it is this bound complex which essentially makes the endogenous IL-2 undetectable by "sandwich" assays. Other cytokines (e.g., IL-1) may be bound to other carrier molecules in serum that effectively masks their detection.
In addition, while various cytokines, including IL-1, have been reported in certain normal or pathological biological fluids, there have been no reports of cytokines or lymphokines in saliva or nasal secretions. In fact, a paper reporting a study of the pharmacodynamics of interferon (Diez, R. A. et al., J. INTERFERON RES. 7: 553-557 (October 1987)) stated that ". . . at present, whether interferon is present in saliva and nasal secretion is unclear."
It would be of great benefit if one could easily, accurately and reproducibly measure the concentrations of various endogenous cytokines in the body fluids. This would create a useful window not only into the immune system but into a myriad of physiologically interacting processes. Such tools would be useful in a variety of settings, allowing the collection of data of importance to basic medical sciences, clinical medicine, epidemiology and the forensic sciences.
What is needed is a reliable method of measuring endogenous cytokines in blood which will result in an accurate blood concentration of the cytokine independent of binding proteins which may be bound to the circulating cytokine. In addition, ability to measure cytokines in a biological fluid such as saliva or nasal secretions would simplify the analysis of an individual's immune system, possibly supplanting the need to perform prolonged, tedious and highly variable assays of these cells' behavior in culture.